Combination Therapy of Human Umbilical Cord BloodCells and Granulocyte Colony Stimulating FactorReduces Histopathological and Motor Impairments in anExperimental Model of Chronic Traumatic Brain InjurySandra A. Acosta1, Naoki Tajiri1, Kazutaka Shinozuka1, Hiroto Ishikawa1, Paul R. Sanberg1,2,
Juan Sanchez-Ramos3,4,5, Shijie Song3,4,5, Yuji Kaneko1, Cesar V. Borlongan1*
1 Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, Florida, United
States of America, 2 Office of Research and Innovation, University of South Florida, Tampa, Florida, United States of America, 3 James Haley Veterans Affairs Medical
Center, Tampa, Florida, United States of America, 4 Department of Neurology, University of South Florida, Tampa, Florida, United States of America, 5 Department of
Molecular Pharmacology and Physiology, University of South Florida, Tampa, Florida, United States of America
Abstract
Traumatic brain injury (TBI) is associated with neuro-inflammation, debilitating sensory-motor deficits, and learning andmemory impairments. Cell-based therapies are currently being investigated in treating neurotrauma due to their ability tosecrete neurotrophic factors and anti-inflammatory cytokines that can regulate the hostile milieu associated with chronicneuroinflammation found in TBI. In tandem, the stimulation and mobilization of endogenous stem/progenitor cells from thebone marrow through granulocyte colony stimulating factor (G-CSF) poses as an attractive therapeutic intervention forchronic TBI. Here, we tested the potential of a combined therapy of human umbilical cord blood cells (hUCB) and G-CSF atthe acute stage of TBI to counteract the progressive secondary effects of chronic TBI using the controlled cortical impactmodel. Four different groups of adult Sprague Dawley rats were treated with saline alone, G-CSF+saline, hUCB+saline orhUCB+G-CSF, 7-days post CCI moderate TBI. Eight weeks after TBI, brains were harvested to analyze hippocampal cell loss,neuroinflammatory response, and neurogenesis by using immunohistochemical techniques. Results revealed that the ratsexposed to TBI treated with saline exhibited widespread neuroinflammation, impaired endogenous neurogenesis in DG andSVZ, and severe hippocampal cell loss. hUCB monotherapy suppressed neuroinflammation, nearly normalized theneurogenesis, and reduced hippocampal cell loss compared to saline alone. G-CSF monotherapy produced partial andshort-lived benefits characterized by low levels of neuroinflammation in striatum, DG, SVZ, and corpus callosum and fornix,a modest neurogenesis, and a moderate reduction of hippocampal cells loss. On the other hand, combined therapy ofhUCB+G-CSF displayed synergistic effects that robustly dampened neuroinflammation, while enhancing endogenousneurogenesis and reducing hippocampal cell loss. Vigorous and long-lasting recovery of motor function accompanied thecombined therapy, which was either moderately or short-lived in the monotherapy conditions. These results suggest thatcombined treatment rather than monotherapy appears optimal for abrogating histophalogical and motor impairments inchronic TBI.
Citation: Acosta SA, Tajiri N, Shinozuka K, Ishikawa H, Sanberg PR, et al. (2014) Combination Therapy of Human Umbilical Cord Blood Cells and GranulocyteColony Stimulating Factor Reduces Histopathological and Motor Impairments in an Experimental Model of Chronic Traumatic Brain Injury. PLoS ONE 9(3): e90953.doi:10.1371/journal.pone.0090953
Editor: Zoran Ivanovic, French Blood Institute, France
Received September 25, 2013; Accepted February 6, 2014; Published March 12, 2014
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone forany lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: Financial support for this study was provided by the Department of Defense W81XWH-11-1-0634, the University of South Florida SignatureInterdisciplinary Program in Neuroscience funds, the University of South Florida and Veterans Administration Reintegration Funds, and the University of SouthFlorida Neuroscience Collaborative Program. CVB and PRS serve as consultant and founder, respectively, of Saneron CCEL Therapeutics, Inc. CVB is funded by theNational Institutes of Health 1R01NS071956-01A1, James and Esther King Biomedical Research Foundation 1KG01-33966, SanBio Inc., KMPHC and NeuralStem Inc.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have read the journal’s policy and have the following conflicts: CVB and PRS serve as consultant and founder, respectively, ofSaneron CCEL Therapeutics, Inc. CVB is supported by National Institutes of Health, National Institute of Neurological Disorders and Stroke 1R01NS071956-01,Department of Defense W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program, SanBio Inc., KMPHC and NeuralStem Inc. CVB isa PLOS ONE Editorial Board member. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. In addition, theopinions expressed in this publication are those of the authors and not of the Department of Veterans Affairs or the US government.
* E-mail: [email protected]
Introduction
Traumatic brain injury (TBI) produces debilitating conditions
that affect millions worldwide [1]. Recent data show that in the
United States alone more than 1.7 millions, including military
personnel, sustain a TBI every year [1,2]. Regardless of the severity
of the trauma, motor, behavioral, intellectual and cognitive
disabilities will be manifested as both short- and long-term
[3,4,5,6,7,8]. In moderate to severe trauma, TBI survivors present
with chronic disabilities associated with loss of primary cerebral
parenchymal tissues, secondary cell death including apoptosis, and
exacerbated neuroinflammation [9,10,11]. Among the worst
outcomes, sensorimotor dysfunctions, massive hippocampal cell
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death, learning and memory impairments, aphasia, anxiety, and
dementia are the most prevalent [12,13,14,15,16]. At present,
clinical treatments are limited and the few that have been utilized
have proven to be ineffective in most of the TBI cases [17,18,19].
Preclinical studies have demonstrated that adult stem/progenitor
cells transplantation is a promising therapeutic intervention for TBI
[20,21]. Bone marrow stromal cells (BMSC), adipose derived stem
cells (ADSC), amniotic fluid stem cells (AFSC) and the mononuclear
fraction of human umbilical cord blood (hUCB) have shown
neuroprotective properties by decreasing inflammation, brain tissue
loss, promoting neurogenesis, and rescuing neurological functions
such as learning and memory in experimental models of chronic
TBI [20,21,22,23,24]. However, the injured micro-environment
limits their regenerative potential [19,25]. For instance, TBI victims
suffer from brain oxygen depletion, vasogenic edema, and
secondary injury signals including reactive oxygen species, exacer-
bated activated MHCII+ cells, astrogliosis and pro-inflammatory
cytokines such as, but not limited to, IL-1beta, TNF-alpha which
can accumulate in the area of injury leading to decreased survival of
transplanted adult stem cells [11,25,26]. The use of combined
therapies stands as a promising technique to overcome molecular
aberrations while enhancing the adult stem cells’ therapeutic
potential in chronic TBI [27,28].
Colony stimulating factors (CSF), also called haemopoietic
growth factors, regulate the mobilization, proliferation, and
differentiation of bone marrow cells. Growth factors such as
granulocyte colony stimulating factor (G-CSF), granulocytes-
macrophages colony stimulating factor (GM-CSF), colony stimu-
lating factor-1(CSF-1), and erythropoietin are currently being
investigated as therapeutics for cancer, certain autoimmune
diseases, ischemic insults and neurodegenerative diseases
[10,29,30]. Recent evidence suggests that G-CSF affords beneficial
effects against central nervous system (CNS) conditions such as
stroke and Alzheimer’s disease [30,31,32]. Short-term treatment of
systemic G-CSF significantly improved cognition accompanied by
reduced central and peripheral inflammation, enhanced neuro-
genesis and decreased the amyloid deposition in the hippocampus
and entorhinal cortex in both mice and rats experimental models
of Alzheimer’s disease [30,32]. Similarly, G-CSF, together with
stem cell factor, restored neural circuits by facilitating anatomical
connections of dendritic spines and branches with the adjacent
infracted area of experimental stroke [31]. Recent clinical trials of
G-CSF treatment in stroke patients have been proven safe [33],
but efficacy remains inconclusive [10].
In the present in vivo study, we assessed if hUCBC transplan-
tation combined with G-CSF treatment afforded enhanced
neuroprotection in a rat CCI model of moderate TBI in the long
term using validated TBI immunohistochemical parameters of
hippocampal cell loss, neuroinflammatory response, and neuro-
genesis. Here we report synergistic effects of hUCB transplantation
and G-CSF treatment as evidenced by highly effective sequestra-
tion of hippocampal cell loss, much more robust reduction in
neuroinflammation and massive neurogenesis in the TBI brain
compared to stand alone therapies.
Methods
SubjectsExperimental procedures were approved by the University of
South Florida Institutional Animal Care and Use Committee
(IACUC). All male rats were housed under normal conditions
(20uC, 50% relative humidity, and a 12-h light/dark cycle).
Normal light/dark cycle was employed. A separate cohort of
animals, saline group, (n = 7); G-CSF group, (n = 8); hUCB group
(n = 8); hUCB+G-CSF (n = 8) underwent the same experimental
above and subjected to behavioral tests to determine the functional
effects of hUCB and G-CSF. All behavioral testing was done
during the light cycle at the same time across testing days.
Necessary precautions were taken to reduce pain and suffering of
animals throughout the study. All studies were performed by
personnel blinded to the treatment condition.
Surgical proceduresTen-week old Sprague–Dawley rats (n = 55) were subjected to
TBI using a controlled cortical impactor (CCI; Pittsburgh
Precision Instruments, Inc, Pittsburgh, PA). Deep anesthesia was
achieved using 1–2% isoflurane in nitrous oxide/oxygen (69/30%)
using a nose mask. All animals were fixed in a stereotaxic frame
(David Kopf Instruments, Tujunga, CA, USA). TBI injury
surgeries consisted of animals subjected to scalp incision to expose
the skull, and craniectomy. An electric drill was used to perform
the craniectomy of about 2.5 mm radius with coordinates
calculated from +0.2 anterior and 20.2 mm lateral right from
bregma [34]. After craniotomy the brain was impacted at the
fronto-parietal cortex with a velocity of 6.0 m/s reaching a depth
of 1.0 mm below the dura matter layer and remained in the brain
for 150 milliseconds (ms). The impactor rod was angled 15udegrees vertically to maintain a perpendicular position in reference
to the tangential plane of the brain curvature at the impact surface.
A linear variable displacement transducer (Macrosensors, Penn-
sauken, NJ), which was connected to the impactor, measured the
velocity and duration to verify consistency. The analgesic
ketoprofen (5 mg kg–1) was administered postoperatively. Rats
were kept under close supervision.
Intravenous administration of G-CSF and hUCB cellsOne week post-TBI CCI surgery, rats were anesthetized with 1–
2% isoflurane in nitrous oxide/oxygen (69/30%) using a nose
mask. Four different groups, (n = 6), of TBI rats were treated
intravenously through the jugular vein with either saline alone
(500 ml of sterile saline), G-CSF (300 mg/kg in 500 ml of sterile
saline), hUCB+saline (46106 viable cells supplied by Saneron
CCEL Therapeutics, Inc., in 500 ml of sterile saline), or hUCB+G-
CSF (46106 viable cells in 500 ml of sterile saline and 300 mg/kg
G-CSF in 500 ml of sterile saline) at 7 days post TBI.
HistologyUnder deep anesthesia, rats were sacrificed 8 weeks after TBI
surgery. For transcardial perfusion, rats were placed on their
backs, and blunt forceps were used to cut through the body cavity.
The opening was extended laterally until reaching the rib cage.
Rib cage was cut, and sternum was lifted to expose the heart. The
heart was held gently, and the needle was inserted J inch into the
left ventricle. The right atrium was cut using the irridectomy
scissors. Through the ascending aorta, approximately 200 ml of
ice cold phosphate buffer saline (PBS) followed by 200 ml of 4%
paraformaldehyde (PFA) in PBS were used for brain perfusion.
Brains were removed and post-fixed in the same fixative at 4uC for
24 hours followed by 30% sucrose in phosphate buffer (PB) for 1
week. Brains were frozen at 224uC, mounted onto a 40 mm
specimen disk using embedding medium. Coronal sectioning was
carried out at a thickness of 40 mm by cryostat. H& E and
Immunostainings were done on every 6th (1/6) coronal section
spanning the frontal cortex and the entire dorsal hippocampus.
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Hematoxylin and eosin analysisHematoxylin and eosin (H&E) staining was performed to
confirm the core impact injury of our TBI model. As shown in our
previous studies [11,35,36,37], we also demonstrated here that the
primary damage produced by the CCI TBI model was to the
fronto-parietal cortex. In addition, H&E staining was analyzed in
the hippocampus to reveal surviving neurons. . In all animals,
sections were anatomically matched. Series of 6 sections per rat
were processed for staining. H&E staining was done on every sixth
coronal section spanning the dorsal hippocampus, starting at
coordinates AP-2.0 mm and ending at AP-3.8 mm from bregma.
Cells presenting with nuclear and cytoplasmic staining (H&E) were
manually counted in the CA3 neurons. CA3 cell counting spanned
the whole CA3 area, starting from the end of hilar neurons to the
beginning of curvature of the CA2 region in both the ipsilateral
and contralateral side. In order to calculate the % of surviving
neurons on the CA3, the % of CA3+ neurons on the ipsilateral
side are compared to the contralateral side to TBI hemispheres.
Sections were examined with Nikon Eclipse 600 microscope at
206 All data are represented as mean values 6SEM, with
statistical significance set at p,0.05.
ImmunohistochemistryStaining for DCX and OX6 was conducted on separate sets of
section of every 6th coronal section throughout the entire striatum
and dorsal hippocampus. In all animals, sections were anatomi-
cally matched. For the DCX staining normal horse serum was
used. For the MHCII (OX6) staining normal goat serum was used.
Sixteen free-floating coronal sections (40 mm) were washed 3 times
in 0.1M phosphate-buffered saline (PBS) to clean the section from
cryoprotectant. Thereafter, all section were incubated in 0.3%
hydrogen peroxide (H2O2) solution for 20 minutes and washed 3
times with PBS for 10 minutes each wash. Next, all sections were
incubated in blocking solution for 1 hour using PBS supplemented
with 3% normal serum and 0.2% Triton X-100. Sections were
then incubated overnight at 4uC with either goat anti rat DCX
(1:150 immature neuronal marker doublecortin or DCX; Santa
Cruz; cat#sc8066), or mouse anti rat MHCII (OX6) (major
histocompatibility complex or MHC class II; 1:750 BD; cat#554926) antibody markers in PBS supplemented with 3% normal
serum and 0.1% triton X-100. Sections were then washed 3 times
with PBS and incubated in biotinylated horse anti-goat secondary
antibody (1:200; Vector Laboratories, Burlingame, CA)for the
DCX staining, or goat anti-mouse goat secondary antibody (1:200;
Vector Laboratories, Burlingame, CA) in PBS supplemented with
normal serum, and 0.1% Triton X-100 for 1 hour. Next, the
sections were incubated for 60 minutes in avidin–biotin substrate
(ABC kit, Vector Laboratories, Burlingame, CA) and washed 3
times with PBS for 10 minute each wash. All sections were then
incubated for 1 minute in 3,30-diaminobenzidine (DAB) solution
(Vector Laboratories) and wash 3 times with PBS for 10 minutes
each wash. Sections were then mounted onto glass slides,
dehydrated in ascending ethanol concentration (70%, 95%, and
100%) for 2 minutes each and 2 minutes in xylenes, then cover-
slipped using mounting medium.
Stereological analysisUnbiased stereology was performed on brain sections immu-
nostained with OX6, and DCX. Sets of 1/6 section, ,16
systematically random sections, of about 240 mm apart, were taken
from the brain spanning AP – 0.2 mm to AP – 3.8 mm in all 24
rats. Activated MHCII+ cells, and differentiation into immature
neurons were visualized by staining with OX6, DCX, respectively.
Positive stains were analyzed with a Nikon Eclipse 600 microscope
and quantified using Stereo Investigator software, version 10
(MicroBrightField, Colchester, VT). The estimated volume of
OX6-positive cells was examined using the Cavalieri estimator
probe of the unbiased stereological cell technique revealing the
volume of OX6 in the cortex, striatum, thalamus, fornix, cerebral
peduncle, and corpus callosum. The literature supports the
concept of the Cavalieri principle as a stereological technique
used to estimate the volume of structures and regions with
arbitrary shape and size, and not only to spherical structures or
regions [35,36,37]. DCX positive cells were counted within the
subgranular zone (SGZ) of the dentate gyrus of the hippocampus,
and within the subventricular zone (SVZ) of the lateral ventricle, in
both hemispheres (ipsilateral and contralateral), using the optical
fractionator probe of unbiased stereological cell counting tech-
nique. The sampling was optimized to count at least 300 cells per
animal with error coefficients less than 0.07. Each counting frame
(1006100 mm for OX6, and DCX) was placed at an intersection
of the lines forming a virtual grid (1756175 mm), which was
randomly generated and placed by the software within the
outlined structure. Section thickness was measured in all counting
sites. For the DCX+ cells analysis in the DG and SVZ, the % of
DCX+ neurons on the ipsilateral side are compared to the
contralateral side to TBI hemispheres.
Behavioral TestsElevated body swing test (EBST) involved handling the animal
by its tail and recording the direction of the swings [38]. The test
apparatus consisted of a clear Plexiglas box (40640635.5 cm).
The animal was gently picked up at the base of the tail, and
elevated by the tail until the animal’s nose is at a height of 2 inches
(5 cm) above the surface. The direction of the swing, either left or
right, was counted once the animals head moves sideways
approximately 10 degrees from the midline position of the body.
After a single swing, the animal was placed back in the Plexiglas
box and allowed to move freely for 30 seconds prior to retesting.
These steps were repeated 20 times for each animal. Normally,
intact rats display a 50% swing bias, that is, the same number of
swings to the left and to the right. A 75% swing bias towards one
direction was used as criterion of TBI motor deficit. For
assessment of motor balance and coordination, the animals were
tested in the rotorod test following the procedures described
elsewhere [39]. The rotorod treadmill (Accuscan, Inc., Columbus,
OH, USA) produced motor balance and coordination data which
were generated by averaging the scores (total time spent on
treadmill divided by 5 trials) for each animal during training and
testing days. Each animal was placed in a neutral position on a
cylinder (3 cm and 1 cm diameter for rats and mice, respectively)
then the rod was rotated with the speed accelerated linearly from
0 rpm to 24 rpm within 60 s, and the time spent on the rotrod was
recorded automatically. The maximum score given to an animal
was fixed to 60. For training, animals were given 5 trials each day
and declared having reached the criterion when they scored 60 in
3 consecutive trials. For testing, animals were given 3 trials and the
average score on these 3 trials was used as the individual rotorod
score.
Statistical analysisFor immunohistochemical data analyses, contralateral and
ipsilateral corresponding brain areas were used as raw data
providing 4 sets of data per treatment condition (TBI+saline alone,
G-CSF+saline, hUCB+G-CSF, or hUCB+saline), therefore two-
way and one-way analysis of variance (ANOVA) was used for
group comparisons, followed by subsequent pairwise comparisons
using post hoc Bonferonni test, which included analyses of any
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differences between treatments, as well as between contralateral
and ipsilateral hemispheres. For behavioral data analyses, repeated
measures of ANOVA and post hoc Bonferroni’s t-tests for each
time point were used to evaluate statistical differences between
treatment groups. For all analyses, differences were considered
significant at p,0.05. All values are expressed as mean6SEM.
Results
Treatment with hUCB and G-CSF monotherapy, andhUCB+G-CSF combined therapy ameliorate TBI–inducedneuroinflammation
The brain regions examined for TBI-induced neuroinflamma-
tion included the gray matter areas of cortex, striatum, thalamus,
SVZ, and DG of the hippocampus, while the white matter areas
analyzed were corpus callosum, fornix, and cerebral peduncle
(Fig. 1A, 2A]. ANOVA revealed overall significant treatment
effects on inflammation as evidenced by OX-6 immunostaining in
all brain regions examined here as follows: cortex F3, 20 = 4.913,
p,0.0001; striatum F3,20 = 6.466, p,0.0001; thalamus
F3,20 = 8.785, p,0.0001; SVZ F3,20 = 6.543, p,0.0001; DG
F3,20 = 4.587, p,0.0001; corpus callosum, F3,20 = 14.6,
p,0.0001; fornix, F3,20 = 9.017, p,0.0001; cerebral peduncle,
F3,20 = 4.638, p,0.0001. Posthoc test analysis revealed a robust
upregulation of activated microglia cells when comparing the total
estimated volume of MHCII+ cells in the ipsilateral hemisphere of
rats exposed to chronic TBI and treated with saline alone to their
contralateral side across all gray and white matter areas analyzed
(p,0.0001), except in the corpus callosum area (p.0.05).
The estimated volume of MHCII+ activated cells was quantified
using stereological techniques. One way ANOVA revealed a
significant treatment effects in the ipsilateral cortex and subcortical
gray matter areas As follows: cortex F3, 20 = 4.869, p,0.0075;
striatum F3,20 = 5.107, p,0.0025; thalamus F3,20 = 7.044,
p,0.0012; SVZ F3,20 = 6.543, p,0.0019; DG F3,20 = 5.237,
p,0.0061. Post hoc Bonferroni’s test revealed that monotherapy
of hUCB cells and the combined therapy of hUCB+G-CSF
significantly decreased the TBI-associated upregulation of
MHCII+ activated cells in the cortex, striatum, thalamus, SVZ,
and DG relative to rats exposed to chronic TBI treated with saline
alone (p,0.05) (Fig. 1B, C). G-CSF monotherapy was also
effective in reducing activated MHCII+ cells in most gray matter
areas analyzed (p,0.05), except in the cortical and thalamic area
compared to treatment of saline alone (p.0.05) (Fig. 1C).
Similar analyses of OX-6 neuroinflammation demonstrated
significant treatment effects in several white matter areas ipsilateral
to TBI injury (Fig. 2A). ANOVA revealed statistical significance in
the following white matter areas as follows: corpus callosum,
F3,20 = 6.506, p,0.0018; fornix, F3,20 = 8.324, p,0.0005; cerebral
peduncle, F3,20 = 4.733, p,0.0088. Post hoc Bonferroni’s test
analysis revealed that combined therapy of hUCB+G-CSF
decreased the TBI-associated upregulation of activated MHCII+cells in corpus callosum, fornix, and cerebral peduncle ipsilateral
to injury when compared to rats exposed to TBI treated with
saline alone (p,0.05) (Fig. 2B, C). hUCB cells alone and G-CSF
alone were effective at significantly suppressing activated MHCII+cells only in two white matter areas, namely the corpus callosum
and fornix relative to rats exposed to chronic TBI treated with
saline alone (p,0.05) (Fig. 2C).
hUCB and G-CSF monotherapy, and combined hUCB+G-CSF attenuate TBI-induced impairment in endogenousneurogenesis
ANOVA revealed significant treatment effects on neurogenesis
in the neurogenic DG (F3, 20 = 9.107, p,0.0005). Post hoc
Bonferroni’s test analysis revealed that hUCB and G-CSF
monotherapy, and the combined therapy of hUCB+G-CSF
significantly enhanced endogenous neurogenesis in the DG in
our model of chronic TBI compared to injured rats treated with
saline alone (p,0.05) (Fig. 3A). Moreover, ANOVA revealed
significant treatment effects on neurogenesis in the other
neurogenic site, SVZ (F3, 20 = 20.00, p,0.0001) (Fig. 3B). Post
hoc Bonferroni’s test analysis revealed a significant upregulation of
neurogenesis in the SVZ of chronically TBI-exposed rats treated
with either hUCB and G-CSF monotherapy, and the combined
therapy of hUCB+G-CSF compared to injured rats treated with
saline alone (p,0.05) (Fig. 3D)
hUCB monotherapy and combined hUCB+G-CSFrobustly, while G-CSF alone moderately decreasehippocampal cell loss in rats exposed to chronic TBI
ANOVA revealed significant treatment effects on TBI-induced
hippocampal cell loss (ANOVA, F3,20 = 159.3, p,0.0001). Treat-
ment with hUCB cell monotherapy and the combined therapy of
hUCB+G-CSF significantly rescued the neuronal cells in the CA3
region of the hippocampus (Fig. 3C). There was a significant
survival of cells in CA3 regions in rats exposed to chronic TBI and
treated with either hUCB cells alone or combined hUCB+G-CSF
compared with injured rats treated with G-CSF alone or saline
(p’s,0.05). Although not as robust an effect as hUCB alone and
combined hUCB+G-GSCF, quantitative analyses also revealed a
significant increase in survival of CA3 neurons in injured rats
treated with G-CSF monotherapy relative to those treated with
saline (p,0.05) (Fig. 3C).
Treatment with hUCB and G-CSF monotherapy, andhUCB+G-CSF combined therapy promote behavioralrecovery in chronic TBI animals
ANOVA revealed main effects of treatment (EBST, p,0.001;
rotorod, p,0.0001) and time (EBST, p,0.001; rotorod,
p,0.0001). ANOVA also revealed treatment by time after TBI
interaction effects in both tasks EBST (F3,27 = 427.11, p,0.0001)
and rotorod test (F3,27 = 564.38, p,0.0001). Within-groups
comparison across weeks revealed performance in EBST and
rotorod test was impaired by moderate TBI immediately after the
injury (Day 0, p,0.05), and remained significantly deficient even
up to the chronic stage (i.e., 56 days post-TBI, p’s,0.05) in TBI-
saline treated animals (Fig. 4). In both tasks, while the TBI-saline
showed a trend of improved recovery over time, their performance
did not reach statistical significance (p’s.0.05) compared to Day 0
post-TBI. In contrast, significant recovery in both EBST and
rotorod were detected in TBI animals that received monotherapy
of hUCB or G-CSF and the combined therapy of hUCB+G-CSF
(Fig. 4A, B). In EBST (Figure 4A), hUCB alone and the combined
hUCB+G-CSF groups displayed significant improvements across
all post-TBI time points (p’s,0.05). In contrast, the monotherapy
of G-CSF while significantly recovered at days 7–14 post-TBI
(p’s,0.05), reverted to Day 0 level at day 28 and day 56 post-TBI
(p’s.0.05). Similarly, TBI-saline treated animals were significantly
impaired throughout the post-TBI time points (p’s,0.05), whereas
TBI animals that received monotherapy of hUCB or G-CSF and
the combined therapy of hUCB+G-CSF displayed significant
improvement across all post-TBI time points (p’s,0.05), and the
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monotherapy of G-CSF only showed temporary recovery at days
7–14 post-TBI (p’s,0.05), then reverting to Day 0 impaired level
at day 28 and day 56 post-TBI (p’s.0.05). Next, between groups
comparisons revealed that the combined therapy of hUCB+G-
CSF displayed the most robust recovery in motor performance
and showing much better improvement over time compared to the
other treatment conditions (p’s,0.05) (Fig. 4A). The monotherapy
of hUCB was able to mimic the motor performance of the
combined hUCB+G-CSF group, but only up to day 28 (p’s,0.05),
with the combined therapy much more improved than the hUCB
monotherapy by day 56 post-TBI (p,0.05). In contrast, the
monotherapy of G-CSF while significantly recovered at days 7–14
compared to TBI-saline group, was still displaying impaired motor
performance compared to monotherapy of hUCB and combined
hUCB+G-CSF group; moreover, the monotherapy of G-CSF was
not statistically different from TBI-saline group on days 28 and 56
(p’s.0.05) (Fig. 4A). Between groups comparisons in the rotorod
tests generally resembled the EBST results, demonstrating the
combined therapy promoted the most effective recovery of motor
balance and coordination as early as day 7 post-TBI which was
maintained, and seemed to display improved recovery over time
(throughout the 56-day post-TBI period) compared to all other
treatment groups (p’s,0.05) (Fig. 4B). The monotherapy of hUCB
exhibited significantly better improvement than G-CSF alone and
TBI-saline throughout the 56-day post-TBI period (p’s,0.05), but
not as fully recovered as the combined therapy group at all-time
points (p’s,0.05) (Fig. 4B). The monotherapy of G-CSF alone
showed a short-lived recovery of motor balance and coordination
at day 7 and day 14 (p’s,0.05), but by day 28 and day 56, this
GCSF alone group did not significantly differ from TBI-saline
(p’s.0.05).
Discussion
This study demonstrates synergistic therapeutic anti-inflamma-
tory potential of combined therapy of hUCB+G-CSF over
monotherapy of hUCB cells or G-CSF in rats exposed to chronic
TBI. Chronic TBI is typically associated with major secondary
molecular injuries whereby chronic neuroinflammation rampantly
instigates an upswing of pro-inflammatory cytokines which further
contribute to neuronal cells death, increase reactive oxygen species
and downregulate endogenous repair mechanism such as neuro-
genesis [11,40]. The involvement of the immune system in the
CNS to either stimulate repair or enhance molecular damage has
become increasingly recognized as a key component of the
pathological onset and progression of many neurological disorders
including TBI and neurodegenerative diseases [41]. We and others
have shown a marked increase in MHCll+ cells in acute and
chronic TBI as well as in many other neuropathological disorders
including Alzheimer’s disease, multiple sclerosis, Parkinson’s
disease, and autoimmune diseases [41,42,43,44]. Increased
expression of MHCII+ cells is directly correlated with neurode-
generation and cognitive declines of these models [43,45]. In
addition, enhanced positive staining for MHCII+ cells is correlated
with the increase of pro-inflammatory cytokines such as TNF-
alpha, IL-6, IL-1 and chemokines such as fractalkine. In contrast,
decreased MHCII+ cells correlate with reduced aberrant accu-
mulation of reactive oxygen species and an increase of anti-
inflammatory cytokines [46,47]. We are cognizant of the need for
cytokine profiling in TBI brain under different treatment
conditions. Currently, clinical treatments to specifically target
the massive inflammatory secondary insults beyond the original
injury, such as chronic white matter injury, are limited and the few
that have been utilized have proven to be ineffective when used in
long-term exposure of chronic TBI [17,18,19].
The present in vivo study sought to examine whether there were
any potential synergistic benefits associated with the use of
combined therapy of hUCB cells along with factors that would
enhance endogenous stem cells such as factor-CSF in a chronic
TBI rat model. We show that combined treatment resulted in
better reduction of neuroinflammation than monotherapy in the
striatum, SVZ and DG, and CC. On the other hand, monother-
apy of hUCB cells or G-CSF reduced neuroinflammation in a few
gray matter areas; monotherapy had little or no effect in the cortex
and thalamus, but worked as well or better in other regions such as
the striatum, SVZ and DG, corpus callosum and fornix, showing
at best modest amelioration of exacerbated MHC+ cells found in
the white matter associated with TBI. Nonetheless, all three
treatments of hUCB alone, G-CSF alone, and combined
hUCB+G-CSF were able to afford decreased hippocampal cell
death, and enhanced neurogenesis in rats exposed to chronic TBI.
These observed histological rescue by hUCB alone, G-CSF alone,
and combined hUCB+G-CSF translated to behavioral recovery,
with the combined therapy affording the most robust improve-
ment in motor performance in treated chronic TBI animals.
These results are in accordance with preclinical data that
suggest that intravenous infusion of hUCB cells in ischemic insults
such as stroke and TBI was able to block neuroinflammation,
improve BBB integrity, as well as decrease brain edema, and
increase endogenous angiogenesis [48,49,50]. Our results demon-
strating a less robust anti-inflammatory effect of G-CSF mono-
therapy concur with reported preclinical data documenting that
the prophylaxis administration of G-CSF alone after brain trauma
has small beneficial effects on motor outcomes, on reducing brain
edema, decreasing cortical contusion volume or modulating glial
cells glutamate concentrations in CSF [51,52]. However, other
studies report that bone marrow mononuclear cells were as
neuroprotective as G-CSF alone in an experimental model of
spinal cord injury [53]. In addition, it has been shown that either
pre-treatment of G-CSF or chronic administration of G-CSF
through mini-pump could in fact be highly beneficial at rescuing
TBI-associated motor lesions, behavioral impairments, cell death
and brain edema [54,55]. Different G-CSF dosing regimens and
disease targets may explain these discrepant results.
In tandem, monotherapy of hUCB cells or G-CSF, and the
combined therapy of hUCB+G-CSF cells, significantly reduced
the TBI-induced loss of pyramidal neuron cells in the CA3 region
of the hippocampus relative to saline alone. Results show that the
Figure 1. Monotherapy of hUCB or G-CSF, and hUCB+G-CSF combined therapy ameliorate TBI–induced neuroinflammation in graymatter areas. Downregulation of activated microglial cells in the ipsilateral side of cortical and subcortical gray matter regions after treatment withhUCB alone, G-CSF alone, and combined hUCB+G-CSF relative to saline. Diagram of a coronal section shows the lesion area in red. The squaresindicate the region of interest for analyses (Fig. 1A). Photomicrographs of gray matter areas of cortex, striatum, thalamus, SVZ and DG of coronalsections from all four groups (Fig. 1B). Arrows indicate positive staining for activated microglia cells. Quantification of OX-6 immunostaining reflectsestimated volume of activated microglia cells of cortex, striatum, thalamus, SVZ, and DG (Fig. 1C). Cortex F3, 20 = 4.913, p,0.0001; striatumF3,20 = 6.466, p,0.0001; thalamus F3,20 = 8.785, p,0.0001; SVZ F3,20 = 6.543, p,0.0001; DG F3,20 = 4.587, p,0.0001. Scale bar in B = 1 mm.* = significant difference between TBI-saline and TBI-G-CSF; & = significant difference between TBI-saline and TBI-hUCB; # = significant differencebetween TBI-saline and TBI-hADSC; ns = no significance. Significance at p’s,0.05.doi:10.1371/journal.pone.0090953.g001
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synergy of the combined therapy is not present since the
monotherapy of hUCB cells equally decreased the neuronal cells
death in this area of the hippocampus relative to saline alone and
monotherapy of G-CSF. Investigations on the neurogenic
potential of present treatments revealed that monotherapy of
hUCB cells or G-CSF, and the combined therapy of hUCB+G-
Figure 2. Monotherapy of hUCB or G-CSF, and hUCB+G-CSF combined therapy ameliorate TBI–induced neuroinflammation in whitematter areas. Downregulation of activated microglia cells in the ipsilateral side of white matter axonal regions after treatment with hUCB alone, G-CSF alone, and combined hUCB+G-CSF relative to TBI-saline. Diagram of a coronal section shows the axonal lesion areas. The squares indicate theregion of interest for analyses (Fig. 2A). Photomicrographs of white matter areas: corpus callosum, fornix, and cerebral peduncle of coronal sectionsfrom all four groups (Fig. 2B). Arrows indicate positive staining for activated microglia cells. Quantification of OX-6 immunostaining reflects estimatedvolume of activated microglia cells in corpus callosum, fornix, and cerebral peduncle (Fig. 2C). Corpus callosum, F3,20 = 14.6, p,0.0001; fornix,F3,20 = 9.017, p,0.0001; cerebral peduncle, F3,20 = 4.638, p,0.0001. Scale bar for B = 1 mm. * = significant difference between TBI-saline and TBI-G-CSF; & = significant difference between TBI-saline and TBI-hUCB; # = significant difference between TBI-saline vs TBI-hADSC; ns = no significance.Significance at p’s,0.05.doi:10.1371/journal.pone.0090953.g002
Combined Therapy of hUCB+G-CSF in Chronic TBI
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CSF cells, equally and significantly increased the estimated total
number of new neurons in the both neurogenic sites of DG and
SVZ relative to saline alone. The influence of hUCB cells or G-
CSF injections on neuronal cell loss and neurogenesis has been
previously reported; hUCB treatments in models of TBI, aging
and stroke studies decrease inflammation and facilitate neurogen-
esis and angiogenesis [56,57]. Likewise, G-CSF treatment has
been shown to be a potent neurogenic modulator in TBI as well as
other neurodegenerative diseases (i.e., hypoxic injury and
Alzheimer’s disease) in that long-term treatments of G-CSF
improve motor function and enhance neurogenesis in the all
neurogenic niches [32,58,59]. The mechanisms responsible for the
Figure 3. hUCB and G-CSF monotherapy, and combined hUCB+G-CSF attenuate TBI-induced impairment in endogenousneurogenesis and cell loss in rats exposed to chronic TBI. Significantly enhanced neural differentiation in the DG and SVZ and increased cellsurvival in CA3 after hUCB or G-CSF monotherapy, and the combined therapy of hUCB+G-CSF relative to saline alone. All 3 treatment conditionspercent neurogenesis in the DG (Fig. 3A). Percent neurogenesis in the SVZ (Fig. 3B). Percentage of neuronal survival in the CA3 region of thehippocampus (Fig. 3C). Photomicrographs of SVZ, DG, and CA3 region (Fig. 3D) Top panel arrows indicate positive staining for neurogenesis in SVZand DG respectivately. Arrow on bottom panel indicates CA3 pyramidal cell loss in TBI-saline (Fig. 3D). F3,20 = 159.3, p,0.0001. Scale bars for FigureD = 50 mm. * = significant difference between TBI-saline and TBI G-CSF; & = significant difference between TBI-saline and TBI-hUCB; # = significantlydifference between TBI-saline and TBI-hADSC; ns = no significance. Significance at p’s,0.05.doi:10.1371/journal.pone.0090953.g003
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beneficial effects of hUCB and G-CSF in the injured brain is not
well elucidated, but may likely involve neurogenesis as demon-
strated in the present study and other reports [60,61,62].
In addition to reducing TBI-mediated histological alterations,
the monotherapy of hUCB and G-CSF and their combined
therapy led to significant behavioral improvements, indicating the
functional benefits of these therapeutics in chronic TBI. That G-
CSF alone was only able to produce short-lived improvements in
motor function suggests that the present drug regimen (single
injection of G-CSF) may need to be optimized, such as repeated
treatments especially during the chronic stage to achieve long-
lasting benefits. Indeed, studies have shown modest behavioral
effects with G-CSF treatments in animal models of neurological
disorders [63,64,65]. On the other hand, hUCB alone seemed to
afford much more improved behavioral outcomes with robust and
stable recovery of motor functions in chronic TBI animals,
indicating that the stem/progenitor cells may be accomplishing a
much more widespread biological action than the drug therapy.
Nonetheless, the combination of G-CSF and hUCB resulted in the
most effective amelioration of TBI-induced behavioral deficits,
suggesting that complementary brain repair processes distinctly or
mutually solicited by these two therapies could have mediated the
Figure 4. Combined hUCB+G-CSF exert robust functional recovery in chronic TBI. A separate cohort of TBI animals, using the sameexperimental paradigm above, was subjected to behavioral tests. Elevated body swing tests revealed that animals displayed normal swing activity(average of 10 swings to both left and right side) prior to TBI (Baseline), but exhibited significant swings to one side after TBI (Day 0 post-TBI)(p’s,0.05 versus Baseline) (Fig. 4A). At post-TBI day 7 and day 14, TBI-hUCB+G-GSF and TBI-hUCB alone promoted significantly better recoverycompared to G-CSF alone, although all three groups performed better than TBI-saline group (a) (p’s,0.05). By day 28, TBI-hUCB and TBI-hUCB+G-CSFwere the only two groups that displayed significant recovery of normal swing activity (p’s,0.05), while TBI-GCSF group reverted to Day 0 post-TBIlevels and did not significantly differ from TBI-saline (Fig. 4A) (p.0.05). By day 56, TBI-hUCB+G-GSF and TBI-hUCB alone were still significantlydisplaying near normal swing activity (p’s,0.05 versus TBI-G-CSF or TBI-saline), but the TBI-hUCB+G-GSF showed significantly better recovery thanTBI-hUCB alone (c) (p,0.05). In addition TBI-GCSF group did not significantly differ from TBI-saline by day 56 (Fig. 4A) (p.0.05). Rotorod testsrevealed that animals learned to balance on the rotating rod (maximum of 60 seconds) prior to TBI (Baseline), but exhibited significant reduction inbalancing time after TBI (Day 0) (p’s,0.05 versus Baseline) (Fig. 4B). At post-TBI day 7 and day 14, the performance in balancing on the rotating rodacross treatment groups showed the following order of best to least recovery: TBI-hUCB+G-GSF.TBI-hUCB alone.G-CSF alone, with all three groupsperforming better than TBI-saline group (a) (p’s,0.05), but the TBI-hUCB+G-CSF displayed the most effective balancing activity at across all timepoints (p’s,0.05 versus all other groups) (Fig. 4B). By day 28 and day 56, only TBI-hUCB and TBI-hUCB+G-CSF were the only two groups that displayedsignificant recovery of balancing activity (p’s,0.05 versus TBI-G-CSF or TBI-saline), while TBI-GCSF group did not significantly differ from TBI-saline(Fig. 4B) (p’s.0.05).doi:10.1371/journal.pone.0090953.g004
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improved behavioral outcome. The mobilization of endogenous
stem cells from the peripheral bone marrow by G-CSF [66,67],
coupled by hUCB grafts secretion of growth factors, as well as a
potential graft-host integration leading to reconstruction of
synaptic circuitry [68,69], may be multi-pronged regenerative
mechanisms triggered by the combined therapy, but not by
monotherapy. Additional studies are warranted to elucidate these
modes of action associated with combined therapy. Furthermore,
the present behavioral tests were limited to motor function,
necessitating that future studies should also consider test of
cognitive performance which is equally altered by TBI.
A potential mechanism of action by which i.v. injected G-CSF
and/or hUCBs influence diverse regions of the brain may be via
receptor-mediated transport and paracrine mechanism. G-CSF is
a cytokine able to readily mobilize stem cells from bone marrow to
the peripheral blood. Previously, it has been shown that these
mobilized cells are able to infiltrate injured tissues promoting self-
repair of neurons, myocytes and other cells. Evidence suggests that
G-CSF can cross the blood brain barrier (BBB) and act upon
neurons and glial through G-CSF receptor. Using radioactive
labeling, an experiment showed that G-CSF is able to pass
through the blood brain barrier of intact animals. The capillaries
associated with the BBB express G-CSF receptor and thus the
entry of G-CSF could be mediated through this receptor [70]. In
our study, gray matter and white matter areas were rescued by the
G-CSF and hUCB monotherapies and more synergistically when
administered concomitantly. Studies have found that activation of
G-CSF receptors on neurons and glial cells downregulates pro-
inflammatory cytokines, and increases neurogenesis, among other
therapeutic effects (e.g., triggers anti-apoptotic pathways and
promotes cerebral angiogenesis), altogether ameliorating sensory
and motor deficits in ischemic injuries [71,72,73,74,75]. In
addition, the combination of G-CSF and hUCB cells can promote
stemness maintenance, and, under appropriate conditions, guide
neural lineage commitment of hUCB in vitro [76,77]. We and
others also recognized the concept of the by-stander effects
whereby mobilized bone marrow cells and hUCB cells in the
periphery stimulate neuroprotection and brain repair by paracrine
mechanism in where cells would secrete trophic factors, growth
factors, chemokines and immune-modulatory cytokines to the
injured milieu [78,79,80,81,82,83]. These studies support our
findings on anti-inflammation, enhanced neurogenesis, and
increased CA3 cell survival in which monotherapy of G-CSF,
hUCB or the combination of both were able to significantly act as
neuroprotective agents in our TBI models.
Taken together, these results indicate that while stand-alone
therapies of hUCB transplantation and G-CSF treatment dem-
onstrated a moderate degree of efficacy, their combination
afforded synergistic robust beneficial effects in neuroinflammation
while decreasing neuronal cell death and stimulating endogenous
neurogenesis in a chronic model of moderate TBI. The combined
therapy also resulted in robust and long-lasting improvements of
motor function. In the clinic, chronic TBI has been visualized as
worsening histopathology with limited therapeutic manipulation
[15,84]. In the present in vivo study, we demonstrated how well
known stand-alone therapies can overcome their own therapeutic
limits in chronic stages of TBI when synergy is achieved through
combination therapy.
Author Contributions
Conceived and designed the experiments: CVB. Performed the exper-
iments: CVB SAA NT HI KS SS. Analyzed the data: CVB SAA.
Contributed reagents/materials/analysis tools: CVB SAA NT HI KS PRS
JSR SS YK. Wrote the paper: CVB SAA NT JSR.
References
1. Faul M, Xu L, Wald MM, Coronado VG (2010) Traumatic brain injury in the
United States: Emergency department visits, hospitalizations and deaths. Atlanta
(GA): Centers for Disease Control and Prevention, National Center for Injury
Prevention and Control
2. Fabrizio KS, Keltner NL (2010) Traumatic brain injury in operation enduring
freedom/operation iraqi freedom: a primer. The Nursing Clinics of North
America 45: 569–580, vi.
3. Ettenhofer KS, Abeles N (2009) The significant of mild traumatic brain injury to
cognition and self-reported sympons in long term recovery from injury. J Clin
Exp Neuropsychol. 31(3):363–72
4. Ozen LJ, Fernandes MA (2012) Slowing down after a mild traumatic brain
injury: a strategy to improve cognitive task performance? Archives of clinical
neuropsychology : the official journal of the National Academy of Neuropsy-
chologists 27: 85–100.
5. Werner C, Engelhard K (2007) Pathophysiology of traumatic brain injury.
British Journal of Anaesthesia 99: 4–9.
6. Bath PM, Sprigg N, England T (2013) Colony stimulating factors (including
erythropoietin, granulocyte colony stimulating factor and analogues) for stroke.
The Cochrane Database of Systematic Reviews 6: CD005207.
7. Trivedi MA, Ward MA, Hess TM, Gale SD, Dempsey RJ, et al. (2007)
Longitudinal changes in global brain volume between 79 and 409 days after
traumatic brain injury: relationship with duration of coma. Journal of
Neurotrauma 24: 766–771.
8. Greenberg G, Mikulis DJ, Ng K, DeSouza D, Green RE (2008) Use of diffusion
tensor imaging to examine subacute white matter injury progression in moderate
to severe traumatic brain injury. Archives of Physical Medicine and
Rehabilitation 89: S45–50.
9. Ng K, Mikulis DJ, Glazer J, Kabani N, Till C, et al. (2008) Magnetic resonance
imaging evidence of progression of subacute brain atrophy in moderate to severe
traumatic brain injury. Archives of Physical Medicine and Rehabilitation 89:
S35–44.
10. Farbota KD, Sodhi A, Bendlin BB, McLaren DG, Xu G, et al. (2012)
Longitudinal volumetric changes following traumatic brain injury: a tensor-
based morphometry study. Journal of the International Neuropsychological
Society18: 1006–1018.
11. Acosta SA, Tajiri N, Shinozuka K, Ishikawa H, Grimmig B, et al. (2013) Long-
term upregulation of inflammation and suppression of cell proliferation in the
brain of adult rats exposed to traumatic brain injury using the controlled cortical
impact model. PLoS One 8: e53376.
12. Smith DH, Nakamura M, McIntosh TK, Wang J, Rodriguez A, et al. (1998)
Brain trauma induces massive hippocampal neuron death linked to a surge in
beta-amyloid levels in mice overexpressing mutant amyloid precursor protein.
The American Journal of Pathology 153: 1005–1010.
13. Coelho CA (2007) Management of discourse deficits following traumatic brain
injury: progress, caveats, and needs. Seminars in Speech and Language 28: 122–
135.
14. Azouvi P, Vallat-Azouvi C, Belmont A (2009) Cognitive deficits after traumatic
coma. Progress in Brain Research 177: 89–110.
15. Bigler ED (2013) Traumatic brain injury, neuroimaging, and neurodegenera-
tion. Frontiers in Human Neuroscience 7: 395.
16. Wong D, Dahm J, Ponsford J (2013) Factor structure of the Depression Anxiety
Stress Scales in individuals with traumatic brain injury. Brain injury 27: 1377–
1382.
17. Cox CS, Jr., Baumgartner JE, Harting MT, Worth LL, Walker PA, et al. (2011)
Autologous bone marrow mononuclear cell therapy for severe traumatic brain
injury in children. Neurosurgery 68: 588–600.
18. Guan J, Zhu Z, Zhao RC, Xiao Z, Wu C, et al. (2013) Transplantation of
human mesenchymal stem cells loaded on collagen scaffolds for the treatment of
9raumatic brain injury in rats. Biomaterials 34: 5937–5946.
19. Walker PA, Shah SK, Jimenez F, Aroom KR, Harting MT, et al. (2012) Bone
marrow-derived stromal cell therapy for traumatic brain injury is neuroprotec-
tive via stimulation of non-neurologic organ systems. Surgery 152: 790–793.
20. Maegele M, Schaefer U (2008) Stem cell-based cellular replacement strategies
following traumatic brain injury (TBI). Minimally Invasive Therapy & Allied
Technologies 17: 119–131.
21. Sanberg PR, Eve DJ, Metcalf C, Borlongan CV (2012) Advantages and
challenges of alternative sources of adult-derived stem cells for brain repair in
stroke. Progress in Brain Research 201: 99–117.
22. Kim HJ, Lee JH, Kim SH (2010) Therapeutic effects of human mesenchymal
stem cells on traumatic brain injury in rats: secretion of neurotrophic factors and
inhibition of apoptosis. Journal of Neurotrauma 27: 131–138.
23. Mahmood A, Lu D, Qu C, Goussev A, Chopp M (2006) Long-term recovery
after bone marrow stromal cell treatment of traumatic brain injury in rats.
Journal of Neurosurgery 104: 272–277.
Combined Therapy of hUCB+G-CSF in Chronic TBI
PLOS ONE | www.plosone.org 10 March 2014 | Volume 9 | Issue 3 | e90953
24. Tajiri N, Acosta S, Glover LE, Bickford PC, Jacotte Simancas A, et al. (2012)
Intravenous grafts of amniotic fluid-derived stem cells induce endogenous cellproliferation and attenuate behavioral deficits in ischemic stroke rats. PLoS One
7: e43779.
25. Tu Y, Chen C, Sun HT, Cheng SX, Liu XZ, et al. (2012) Combination of
temperature-sensitive stem cells and mild hypothermia: a new potential therapyfor severe traumatic brain injury. Journal of Neurotrauma 29: 2393–2403.
26. Ghirnikar RS, Lee YL, Eng LF (1998) Inflammation in traumatic brain injury:role of cytokines and chemokines. Neurochemical Research 23: 329–340.
27. Campbell K, Knuckey NW, Brookes LM, Meloni BP (2013) Efficacy of mildhypothermia (35 degrees C) and moderate hypothermia (33 degrees C) with and
without magnesium when administered 30 min post-reperfusion after 90 min ofmiddle cerebral artery occlusion in Spontaneously Hypertensive rats. Brain
Research 1502: 47–54.
28. Mahmood A, Wu H, Qu C, Xiong Y, Chopp M (2013) Effects of treating
traumatic brain injury with collagen scaffolds and human bone marrow stromalcells on sprouting of corticospinal tract axons into the denervated side of the
spinal cord. Journal of Neurosurgery 118: 381–389.
29. Lyman GH, Dale DC, Culakova E, Poniewierski MS, Wolff DA, et al. (2013)
The impact of the granulocyte colony-stimulating factor on chemotherapy doseintensity and cancer survival: a systematic review and meta-analysis of
randomized controlled trials. Annals of Oncology 24: 2475–2484.
30. Sanchez-Ramos J, Song S, Sava V, Catlow B, Lin X, et al. (2009) Granulocyte
colony stimulating factor decreases brain amyloid burden and reverses cognitive
impairment in Alzheimer’s mice. Neuroscience 163: 55–72.
31. Cui L, Murikinati SR, Wang D, Zhang X, Duan WM, et al. (2013)Reestablishing neuronal networks in the aged brain by stem cell factor and
granulocyte-colony stimulating factor in a mouse model of chronic stroke. PLoS
One 8: e64684.
32. Prakash A, Medhi B, Chopra K (2013) Granulocyte colony stimulating factor
(GCSF) improves memory and neurobehavior in an amyloid-beta inducedexperimental model of Alzheimer’s disease. Pharmacology, Biochemistry, and
Behavior 110: 46–57.
33. England TJ, Abaei M, Auer DP, Lowe J, Jones DR, et al. (2012) Granulocyte-
colony stimulating factor for mobilizing bone marrow stem cells in subacutestroke: the stem cell trial of recovery enhancement after stroke 2 randomized
controlled trial. Stroke 43: 405–411.
34. Paxinos G, Watson C (2005) The rat brain in stereotaxic coordinates. 5th ed.
San Diego, CA: Academic Press.
35. Glover LE, Tajiri N, Lau T, Kaneko Y, van Loveren H, et al. (2012) Immediate,
but not delayed, microsurgical skull reconstruction exacerbates brain damage inexperimental traumatic brain injury model. PLoS One 7: e33646.
36. Liu CY (2008) Combined therapies: National Institute of Neurological Disorders
and Stroke funding opportunity in traumatic brain injury research. Neurosur-
gery 63: N12.
37. Yu S, Kaneko Y, Bae E, Stahl CE, Wang Y, et al. (2009) Severity of controlled
cortical impact traumatic brain injury in rats and mice dictates degree ofbehavioral deficits. Brain Research 1287: 157–163.
38. Borlongan CV, Sanberg PR (1995) Elevated body swing test: a new behavioral
parameter for rats with 6-hydroxydopamine-induced hemiparkinsonism. The
Journal of neuroscience 15: 5372–5378.
39. Takahashi K, Yasuhara T, Shingo T, Muraoka K, Kameda M, et al. (2008)Embryonic neural stem cells transplanted in middle cerebral artery occlusion
model of rats demonstrated potent therapeutic effects, compared to adult neural
stem cells. Brain Research 1234: 172–182.
40. Frugier T, Morganti-Kossmann MC, O’Reilly D, McLean CA (2010) In situ
detection of inflammatory mediators in post mortem human brain tissue aftertraumatic injury. Journal of Neurotrauma 27: 497–507.
41. Hernandez-Ontiveros DG, Tajiri N, Acosta S, Giunta B, Tan J, et al. (2013)
Microglia activation as a biomarker for traumatic brain injury. Frontiers in
Neurology 4: 30.
42. Acosta SA, Tajiri N, Shinozuka K, Ishikawa H, Grimmig B, et al. (2013) Long-
term upregulation of inflammation and suppression of cell proliferation in thebrain of adult rats exposed to traumatic brain injury using the controlled cortical
impact model. PLoS One 8: e53376.
43. Yasuda Y, Shinagawa R, Yamada M, Mori T, Tateishi N, et al. (2007) Long-
lasting reactive changes observed in microglia in the striatal and substantia nigralof mice after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Brain Research
1138: 196–202.
44. Imamura K, Hishikawa N, Ono K, Suzuki H, Sawada M, et al. (2005) Cytokine
production of activated microglia and decrease in neurotrophic factors ofneurons in the hippocampus of Lewy body disease brains. Acta Neuropatho-
logica 109: 141–150.
45. Sasaki A, Kawarabayashi T, Murakami T, Matsubara E, Ikeda M, et al. (2008)
Microglial activation in brain lesions with tau deposits: comparison of humantauopathies and tau transgenic mice TgTauP301L. Brain Research 1214: 159–
168.
46. Bachstetter AD, Rowe RK, Kaneko M, Goulding D, Lifshitz J, et al. (2013) The
p38alpha MAPK regulates microglial responsiveness to diffuse traumatic braininjury. The Journal of Neuroscience 33: 6143–6153.
47. Imamura K, Hishikawa N, Sawada M, Nagatsu T, Yoshida M, et al. (2003)Distribution of major histocompatibility complex class II-positive microglia and
cytokine profile of Parkinson’s disease brains. Acta Neuropathologica 106: 518–526.
48. Lu D, Sanberg PR, Mahmood A, Li Y, Wang L, et al. (2002) Intravenous
administration of human umbilical cord blood reduces neurological deficit in therat after traumatic brain injury. Cell Transplantation 11: 275–281.
49. Taguchi A, Soma T, Tanaka H, Kanda T, Nishimura H, et al. (2004)Administration of CD34+ cells after stroke enhances neurogenesis via
angiogenesis in a mouse model. The Journal of Clinical Investigation 114:330–338.
50. Huang X, Zhang Y, Li S, Tang Q, Wang Z, et al. (2013) IntracerebroventricularTransplantation of ex vivo Expanded Endothelial Colony-Forming Cells
Restores Blood Brain Barrier Integrity and Promotes Angiogenesis of Micewith Traumatic Brain Injury. Journal of Neurotrauma 30:2080–2088.
51. Sakowitz OW, Schardt C, Neher M, Stover JF, Unterberg AW, et al. (2006)Granulocyte colony-stimulating factor does not affect contusion size, brain
edema or cerebrospinal fluid glutamate concentrations in rats followingcontrolled cortical impact. Acta Neurochirurgica Supplement 96: 139–143.
52. Sheibani N, Grabowski EF, Schoenfeld DA, Whalen MJ (2004) Effect ofgranulocyte colony-stimulating factor on functional and histopathologic outcome
after traumatic brain injury in mice. Critical Care Medicine 32: 2274–2278.
53. Guo X, Bu X, Li Z, Yan Z, Jiang J, et al. (2012) Comparison of autologous bone
marrow mononuclear cells transplantation and mobilization by granulocytecolony-stimulating factor in experimental spinal injury. The International
Journal of Neuroscience 122: 723–733.
54. Yang DY, Chen YJ, Wang MF, Pan HC, Chen SY, et al. (2010) Granulocyte
colony-stimulating factor enhances cellular proliferation and motor function
recovery on rats subjected to traumatic brain injury. Neurological Research 32:1041–1049.
55. Khatibi NH, Jadhav V, Saidi M, Chen W, Martin R, et al. (2011) Granulocyte
colony-stimulating factor treatment provides neuroprotection in surgically
induced brain injured mice. Acta Neurochirurgica Supplement 111: 265–269.
56. Iskander A, Knight RA, Zhang ZG, Ewing JR, Shankar A, et al. (2013)
Intravenous administration of human umbilical cord blood-derived AC133+endothelial progenitor cells in rat stroke model reduces infarct volume: magnetic
resonance imaging and histological findings. Stem Cells Translational Medicine2: 703–714.
57. Shahaduzzaman M, Golden JE, Green S, Gronda AE, Adrien E, et al. (2013) Asingle administration of human umbilical cord blood T cells produces long-
lasting effects in the aging hippocampus. Age 35: 2071–2087.
58. Popa-Wagner A, Stocker K, Balseanu AT, Rogalewski A, Diederich K, et al.
(2010) Effects of granulocyte-colony stimulating factor after stroke in aged rats.Stroke 41: 1027–1031.
59. Yang YN, Lin CS, Yang CH, Lai YH, Wu PL, et al. (2013) NeurogenesisRecovery Induced by Granulocyte-Colony Stimulating Factor in Neonatal Rat
Brain after Perinatal Hypoxia. Pediatrics and Neonatology. 54:380–388.
60. Jung KH, Chu K, Lee ST, Kim SJ, Sinn DI, et al. (2006) Granulocyte colony-
stimulating factor stimulates neurogenesis via vascular endothelial growth factorwith STAT activation. Brain Research 1073–1074: 190–201.
61. Minnerup J, Sevimli S, Schabitz WR (2009) Granulocyte-colony stimulatingfactor for stroke treatment: mechanisms of action and efficacy in preclinical
studies. Experimental & Translational Stroke Medicine 1: 2.
62. Zhao LR, Piao CS, Murikinati SR, Gonzalez-Toledo ME (2013) The role of
stem cell factor and granulocyte-colony stimulating factor in treatment of stroke.Recent patents on CNS Drug Discovery 8: 2–12.
63. Maurer MH, Schabitz WR, Schneider A (2008) Old friends in new
constellations–the hematopoetic growth factors G-CSF, GM-CSF, and EPO
for the treatment of neurological diseases. Current Medicinal Chemistry 15:1407–1411.
64. Bakhtiary M, Marzban M, Mehdizadeh M, Joghataei MT, Khoei S, et al. (2010)Comparison of transplantation of bone marrow stromal cells (BMSC) and stem
cell mobilization by granulocyte colony stimulating factor after traumatic braininjury in rat. Iranian Biomedical Journal 14: 142–149.
65. Pereira Lopes FR, Martin PK, Frattini F, Biancalana A, Almeida FM, et al.(2013) Double gene therapy with granulocyte colony-stimulating factor and
vascular endothelial growth factor acts synergistically to improve nerveregeneration and functional outcome after sciatic nerve injury in mice.
Neuroscience 230: 184–197.
66. Joo YD, Lee WS, Won HJ, Lee SM, Choi JH, et al. (2011) Upregulation of
TLR2 expression on G-CSF-mobilized peripheral blood stem cells is responsiblefor their rapid engraftment after allogeneic hematopoietic stem cell transplan-
tation. Cytokine 54: 36–42.
67. Loving CL, Kehrli ME Jr, Brockmeier SL, Bayles DO, Michael DD, et al. (2013)
Porcine granulocyte-colony stimulating factor (G-CSF) delivered via replication-
defective adenovirus induces a sustained increase in circulating peripheral bloodneutrophils. Biologicals 41:368–376.
68. Dasari VR, Veeravalli KK, Saving KL, Gujrati M, Fassett D, et al. (2008)
Neuroprotection by cord blood stem cells against glutamate-induced apoptosis is
mediated by Akt pathway. Neurobiology of Disease 32: 486–498.
69. Willing AE, Vendrame M, Mallery J, Cassady CJ, Davis CD, et al. (2003)Mobilized peripheral blood cells administered intravenously produce functional
recovery in stroke. Cell Transplantation 12: 449–454.
70. Zhao LR, Navalitloha Y, Singhal S, Mehta J, Piao CS, et al. (2007)
Hematopoietic growth factors pass through the blood-brain barrier in intact
rats. Experimental Neurology 204: 569–573.
71. Schneider A, Kruger C, Steigleder T, Weber D, Pitzer C, et al. (2005) Thehematopoietic factor G-CSF is a neuronal ligand that counteracts programmed
Combined Therapy of hUCB+G-CSF in Chronic TBI
PLOS ONE | www.plosone.org 11 March 2014 | Volume 9 | Issue 3 | e90953
cell death and drives neurogenesis. Journal of Clinical Investigation 115: 2083–
2098.72. Toth ZE, Leker RR, Shahar T, Pastorino S, Szalayova I, et al. (2008) The
combination of granulocyte colony-stimulating factor and stem cell factor
significantly increases the number of bone marrow-derived endothelial cells inbrains of mice following cerebral ischemia. Blood 111: 5544–5552.
73. Shyu WC, Lin SZ, Yang HI, Tzeng YS, Pang CY, et al. (2004) Functionalrecovery of stroke rats induced by granulocyte colony-stimulating factor-
stimulated stem cells. Circulation 110: 1847–1854.
74. Hartung T (1998) Anti-inflammatory effects of granulocyte colony-stimulatingfactor. Current Opinion in Hematology 5: 221–225.
75. Morita Y, Takizawa S, Kamiguchi H, Uesugi T, Kawada H, et al. (2007)Administration of hematopoietic cytokines increases the expression of anti-
inflammatory cytokine (IL-10) mRNA in the subacute phase after stroke.Neuroscience Research 58: 356–360.
76. Tsuji T, Nishimura-Morita Y, Watanabe Y, Hirano D, Nakanishi S, et al. (1999)
A murine stromal cell line promotes the expansion of CD34high+-primitiveprogenitor cells isolated from human umbilical cord blood in combination with
human cytokines. Growth Factors 16: 225–240.77. Stachura DL, Svoboda O, Campbell CA, Espin-Palazon R, Lau RP, et al. (2013)
The zebrafish granulocyte colony stimulating factors (Gcsfs): two paralogous
cytokines and their roles in hematopoietic development and maintenance.Blood.
78. Borlongan CV, Hadman M, Sanberg CD, Sanberg PR (2004) Central nervous
system entry of peripherally injected umbilical cord blood cells is not required for
neuroprotection in stroke. Stroke 35: 2385–2389.
79. Borlongan CV (2011) Bone marrow stem cell mobilization in stroke: a
‘bonehead’ may be good after all! Leukemia 25: 1674–1686.
80. Massengale M, Wagers AJ, Vogel H, Weissman IL (2005) Hematopoietic cells
maintain hematopoietic fates upon entering the brain. Journal of Experimental
Medicine 201: 1579–1589.
81. Zhang C, Li Y, Chen J, Gao Q, Zacharek A, et al. (2006) Bone marrow stromal
cells upregulate expression of bone morphogenetic proteins 2 and 4, gap
junction protein connexin-43 and synaptophysin after stroke in rats. Neurosci-
ence 141: 687–695.
82. Parr AM, Tator CH, Keating A (2007) Bone marrow-derived mesenchymal
stromal cells for the repair of central nervous system injury. Bone Marrow
Transplantation 40: 609–619.
83. Yang M, Wei X, Li J, Heine LA, Rosenwasser R, et al. (2010) Changes in host
blood factors and brain glia accompanying the functional recovery after systemic
administration of bone marrow stem cells in ischemic stroke rats. Cell
Transplantation 19: 1073–1084.
84. Lewis FD, Horn GJ (2013) Traumatic brain injury: analysis of functional deficits
and posthospital rehabilitation outcomes. Journal Special Operations Medicine
13: 56–61.
Combined Therapy of hUCB+G-CSF in Chronic TBI
PLOS ONE | www.plosone.org 12 March 2014 | Volume 9 | Issue 3 | e90953
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